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The limitations o f the study have been discussed above in terms o f improvements to the procedure but there are also more general issues about the design o f stable iron isotope studies that potentially limit the interpretation o f the results. As referred to in section 4.3.2, it seems likely that single meal studies do not adequately characterise iron bioavailability in adult subjects in the longer term. The study described here was a single meal study, it must be acknowledged that the interpretation o f any results would be affected by this point. However, given the difficulties o f conducting whole diet studies in young children, single meal studies remain a legitimate research option. Another issue is that this method does not give information about the absolute

amounts o f iron absorbed. Unlike radioactive iron isotopes, stable isotopes are

enrich the natural pool o f stable iron isotopes to a measurable level, resulting in an increase o f the total amount o f iron consumed. This type o f experiment does however estimate the relative effects o f diet on iron bioavailability. This is in fact highly useful information as it can be translated into practical, evidenced-based dietary advice on how to maximise non-haem iron absorption from the diet.

A further issue is a theoretical possibility that extrinsic stable iron isotope labels do not adequately represent the iron absorption process. Extrinsic labels are added to foods immediately prior to consumption whereas intrinsic labels are biologically incorporated into plant or animal tissue by addition to growing medium or injection into young animals. Extrinsic labels are obviously a more practical option in this type o f research. Radioactive iron extrinsic labels have been shown, in adult subjects, to label the non-haem pool as effectively as intrinsic labels (Hallberg & Bjom- Rasmussen 1972, Bjom-Rasmussen et al 1972, Cook et al 1972). However, stable iron isotopes may function differently as, unlike radioactive isotopes, they have to be given in some quantity and cannot be regarded as massless. In the only study o f its type, Boza et al (1995) found that in a rat model percentage retention o f intrinsic stable iron isotope was significantly lower than from extrinsic stable iron isotope (p<0.01). They concluded that the differences were small enough not to invalidate the extrinsic labelling method but the authors suggested that results o f studies using extrinsic stable isotopes should be interpreted cautiously.

As indicated above, the iron isotopes have to be given in doses rather greater than the native iron in test meals (certainly the case for children). One might speculate that adding relatively large isotope doses o f iron to the native iron must have some effect on percentage absorption as adult subjects show a decrease in percentage absorption with increasing amounts o f iron (Bothwell et al 1979). It seems possible that the present study, had reliable data been obtained, would have underestimated the

percentage o f non-haem iron absorbed from the test meals. However these

observations in adult subjects are from pharmaceutical doses o f iron. Bezwoda and colleagues (1983) found that increasing the amount o f non-haem iron in a meal from 1.52mg to 5.72mg resulted in a decrease in average percentage absorption o f non- haem iron from 18.0% to 6.4% but the absolute amounts o f non-haem iron absorbed were very similar. Similar studies have not been carried in child subjects. However,

Bezwoda and colleagues work (1983) suggests that the absolute amounts o f iron absorbed at higher levels o f iron intake will be similar to that seen at lower intakes. Interestingly, 1.52mg and 5.72mg approximate the amount o f iron in test meals used in Study 2 and the amount in the meals plus the isotope dose respectively. The important question from the point o f view o f giving effective dietary advice is simply if drinking milk with a meal is related to a relative decrease in the amount o f iron absorbed and this study design would have provided an answer to this question.

One o f the study limitations was the poor response rate to the invitation to participate. This is no doubt due to the involved protocol and requirement for the child subjects to provide two blood samples. The most obvious disadvantage is that this prolonged the study. However, there could be a further disadvantage. It is often the case that the few families who are willing to let their children participate in an invasive study are unrepresentative o f the general population. This raises the possibility that study results (had there been any) would not have applicable to the wider population o f

children. However, the study was measuring the outcome o f a physiological

mechanism ie iron absorption and it would be reasonable to expect healthy children to have the same iron absorbing mechanisms. It is also the case that factors such as social class (see Appendix 3) are associated with poor iron status. As the degree o f body iron depletion is one o f the strongest influences on iron absorption (Fairweather- Tait 1995) the net result is that social class could be a confounding factor when interpreting iron bioavailability data. On a practical note this could be avoided by recruiting children who have red blood cell indices and ferritin levels within the normal range.

It has been reported that iron absorption at the level o f the enterocyte is regulated, in part, by the amount o f iron recently consumed in the diet (Lieu et al 2001). It is therefore reasonable to speculate that the test meal given on Day 1 o f this study might influence the iron bioavailability o f the test meal on Day 2. For example, if a subject consumed a test meal without milk on day 1, the relatively high iron content o f the meal might cause a reduced amount o f iron to be absorbed on Day 2 (test meal plus milk). It is possible that this effect would be ascribed to the presence o f milk when in fact it was the influence o f the previous day’s intake. In practice the subjects were randomly allocated to receive either milk or water on the first day to account for this

effect. However, it must be acknowledged that this have could limit the interpretation o f the findings (had they been obtained). Note that while all the participating children were breastfed, they were fed for varying lengths o f time and it is not possible to assess what the influence o f breastfeeding, if any, were on any potential results. It is possible that receiving breastmilk rather than iron fortified formulae could be one o f the reasons why these children met the inclusion criteria o f haemoglobin concentration 9.5-11.5g/dl, that is, they were less iron replete.